(1-3)-beta-D glucan molecule detection method based on surface enhanced Raman scattering technology

Abstract

The invention discloses a surface enhanced Raman scattering technology-based (1-3)-beta-D glucan molecule detection method, which comprises the following steps of: obtaining a flexible gold (Au) nano finger array structure on a silicon substrate through nano imprinting; immersing the flexible Au nano finger array structure into an antibody solution corresponding to (1-3)-beta-D-glucan for incubation; the flexible Au fingers are taken out and naturally dried, the flexible Au fingers collapse mutually under the action of surface tension in the liquid volatilization process, and a tetramer Au nano finger / antibody / Au nano finger coupling structure is formed; and finally, the structure is built in the groove of the biological glass slide, so that the chip for detecting the (1-3)-beta-D-glucan by the SERS is completed. The method disclosed by the invention is simple, rapid and sensitive, and is suitable for rapidly detecting whether fungal infection exists in serum or tear of a patient on site without waiting for secondary treatment.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a method for detecting (1-3)-β-D-glucan molecules based on surface-enhanced Raman scattering technology. Background Technology

[0002] Invasive fungal infections (IFIs) are systemic infections caused by opportunistic pathogens (such as Candida, Aspergillus, and Cryptococcus) or endemic pathogens, characterized by tissue invasion and dissemination. They are common in immunocompromised hosts, including those with hematologic malignancies, solid organ transplant recipients, those undergoing long-term glucocorticoid therapy, and those with acquired immunodeficiency syndrome (AIDS). Over the past decade, IFIs have become a focus of global public health concern. Fungi infect billions of people annually, most of which are relatively mild, but some severe fungal infections cause millions of deaths, comparable to those from tuberculosis or malaria. Besides detecting invasive fungi in patient serum, they are also frequently found in the tear fluid of the eye. For these patients, it is crucial to first confirm an invasive fungal infection before initiating antibiotic treatment. Therefore, accurate diagnosis of invasive fungal infections is a vital part of treatment.

[0003] Invasive fungi are composed of a variety of molecules, among which (1-3)-β-D-glucan is a characteristic fungal cell wall component, and its discovery provides a new direction for the potential monitoring and diagnosis of invasive fungal infections. Fungal cell walls have long been considered static exoskeletal structures serving only a mechanical support function; however, recent research evidence suggests that they are essentially highly dynamic functional barriers. For most fungi, the structural core polysaccharides of the cell wall are mainly composed of glucan, chitin, and mannan, with glucan being the most important and abundant polysaccharide component in most fungal cell walls, typically accounting for 35% to 60% of the total cell wall weight. The glucan component is mainly composed of glucose polymers, these glucose units are linearly arranged in a β-configuration via glycosidic bonds between carbon 1 and carbon 3, forming a (1-3)-β-D-glucan backbone. The biosynthesis of this polysaccharide backbone involves the transport of glucose subunits, which are first transported to the plasma membrane, then transported across the membrane, and arranged via linear β(1-3) glycosidic bonds. The key enzyme responsible for forming this polysaccharide backbone is (1-3)-β-D-glucan synthase. The polysaccharide backbone typically consists of approximately 1500 glucose subunits, but within each chain, branches are formed via (1-4) or (1-6) glycosidic bonds. The distribution of branches is highly variable and species-specific. This structural diversity reflects the differences in environmental adaptations among different fungi during evolution. When (1-3)-β-D-glucan is integrated into the fungal cell wall, it is usually present as an insoluble structure; however, in the presence of blood or other bodily fluids, (1-3)-β-D-glucan transforms into single-helical, triple-helical (most common), or random helical shapes and becomes soluble. This soluble (1-3)-β-D-glucan may modulate the immune system by inhibiting phagocytosis by leukocytes. Therefore, the detection of (1-3)-β-D-glucan can be used to aid in the diagnosis of invasive fungal infections and to monitor response to treatment.

[0004] Currently, ELISA fluorescence luminescence is the primary clinical method for measuring collected serum or tears. This involves centrifuging and lysing the collected serum or tears to release (1-3)-β-D-glucan from the fungi, which is then indirectly detected using biolabeled fluorescence. However, unlike the detection of other molecules in serum, ELISA fluorescence luminescence typically requires 24 hours. This 24-hour period allows for changes in the fungal flora, making it impossible to determine the current state of the fungal infection and thus affecting treatment outcomes. Furthermore, for ophthalmic fungal infections, the 24-hour result time prevents immediate medication dispensing, necessitating secondary visits and significantly impacting patient convenience. Additionally, the indirect nature of clinical ELISA fluorescence luminescence diagnosis can lead to missed diagnoses, further affecting clinical treatment. Therefore, the development and application of powerful, rapid, simple, and low-cost diagnostic methods are urgently needed.

[0005] Surface-enhanced Raman scattering (SERS) can achieve single-molecule-level fingerprint detection by combining the direct detection of molecular fingerprint features with light field enhancement, and has important application prospects in the molecular detection of medical disease biomarkers. Importantly, molecular fingerprinting and rapid (second-level) speed are particularly useful for some time-critical molecular diagnostics. However, the current use of SERS for the detection of (1-3)-β-D-glucan molecules, biomarkers of invasive fungal infections in the serum or tears of clinical patients, faces more significant challenges in terms of detection strategy: (1) Liquid serum or tears must be used as the test sample, and washing processes should not be introduced to ensure accurate quantitative detection of biomarker molecules in serum or tears. (2) In addition to (1-3)-β-D-glucan molecules, there are thousands of other known and unknown molecules in serum or tears. How to detect only (1-3)-β-D-glucan molecules in serum or tears without being affected by other molecules is a challenge. If serum or tears are used as direct detection samples, the SERS fingerprint spectrum obtained is a mixed spectrum, which can no longer identify (1-3)-β-D-glucan molecules in serum or tears through fingerprint. (3) How to achieve accurate quantitative detection of (1-3)-β-D-glucan marker molecules in liquid serum or tears? Due to the poor uniformity of local light field hotspots of photonic chips prepared by photolithography, electron beam etching, etc., and the fact that the spectral intensity is easily affected by multiple factors such as desorption of marker molecules in liquid serum, laser thermal effect and optical operation, the intensity of the Raman fingerprint peak of the analyte fluctuates greatly, making accurate quantification impossible. High specificity detection requires that the photonic chip can capture marker molecules, high sensitivity detection requires that the captured marker is in the enhanced coupling electromagnetic field region, and quantitative detection requires the introduction of internal standard molecules. The simultaneous realization of these three is the key to the detection of (1-3)-β-D-glucan molecules in SERS serum or tears.

[0006] In our previous work, we combined photolithography and nanoimprinting techniques to achieve an ordered structure of flexible metal nanofinger arrays with precisely tunable structural units (ACS Nano, 2017, 11(6):5836−5843; Small, 2018, 14:1801146−1801155; ZL201710825180.0; ZL201910666854.6). This metal nanofinger array can be used to collapse and bond together between closely spaced flexible nanofingers by titrating a solvent and utilizing the surface tension during the solvent's evaporation process. This technology allows for the pre-attachment of (1-3)-β-D-glucan antibody modification technology to the Au surface. Through collapse, these antibodies are then brought together to form an Au nanofinger / antibody / Au nanofinger coupling structure. Since the spatial longitudinal dimension of the (1-3)-β-D-glucan aptamer is in the sub-nanometer range, the resulting coupling structure can achieve enhanced electromagnetic fields under quantum plasmon resonance modulation. In this coupling structure, the antibody molecule acts as a gap layer, which can both capture and locally store the (1-3)-β-D-glucan marker. Because the enhanced electromagnetic field is mainly distributed within the gap layer, highly selective and sensitive SERS detection of (1-3)-β-D-glucan molecules can be achieved simultaneously. In practical applications, by dripping serum or tears into a nanofinger coupling structure substrate with a pre-embedded biocarrier sheet, highly selective and sensitive measurement of specific biomarker molecules can be achieved in a liquid serum or tear environment. Since the capture time is about 15 minutes and the detection time is about 1 second, the total detection time of 15 minutes is much shorter than the current hospital standard detection time of 24 hours for (1-3)-β-D-glucan molecules. Summary of the Invention

[0007] Objective of the invention: To provide a detection method for the highly selective, highly sensitive and rapid detection of (1-3)-β-D-glucan molecules, a disease marker for invasive fungal infections in liquid serum or tears. This method requires a simple and reasonable substrate preparation, and can be prepared and stored in large quantities at low cost, which can greatly shorten the detection time of (1-3)-β-D-glucan molecules.

[0008] To achieve the above objectives, the present invention employs the following detection technology solution:

[0009] A method for detecting (1-3)-β-D-glucan molecules based on surface-enhanced Raman scattering technology includes the following steps:

[0010] Step 1: On a silicon substrate, a flexible Au nanofinger ordered array structure is prepared by nanoimprinting, Au thin film deposition and reactive ion beam etching, wherein the finger pillars are made of UV imprinting adhesive and Au layer is deposited on top;

[0011] Step 2: The ordered array structure of flexible Au nanofinger obtained in Step 1 was immersed in the antibody solution corresponding to (1-3)-β-D-glucan and incubated in a refrigerator at 4 ℃ for 12 h, so that the antibody molecules in the solution were adsorbed and connected to the Au surface. After the incubation time was over, it was taken out and dried. During the drying process, the micro-surface tension generated by solvent evaporation caused the Au nanofingers to collapse and stick together, forming a tetrameric Au nanofinger / antibody / Au nanofinger coupling structure based on antibody molecules as gaps. The antibody molecules are distributed on the surface of the Au layer, and finally a molecular capture system is formed. The above incubation process was repeated twice.

[0012] Step 3: Implant the tetrameric Au nanofinger / antibody / Au nanofinger coupling structure obtained in Step 2 into the biocarrier chip to form a detection chip;

[0013] Step 4: Fill the tetrameric Au nanofinger / antibody / Au nanofinger coupled structure detection chip obtained in Step 3 with filtered and lysed complex serum and tears containing (1-3)-β-D-glucan, and let it stand for 15 minutes or more.

[0014] Step 5: Raman measurement is performed on the detection chip filled with liquid serum or tears from Step 4. The Raman spectrometer is focused onto the chip surface in the liquid, the excitation wavelength is 785 nm, and the time is 1 second. A bimolecular Raman spectrum containing only (1-3)-β-D-glucan and its corresponding antibody is obtained. Specific identification is performed through the fingerprint characteristic peaks of both, and the results are obtained through the 1134 cm⁻¹ peak. -1 and 1065cm -1 The Raman peak intensity ratio is used as the standard for quantitative detection.

[0015] The surface-enhanced Raman substrate preparation method described in step 1, wherein the height of a single nanofinger in the ordered array structure of Au nanofingers is 350 nanometers, including a 300-nanometer polymer pillar and a top 50-nanometer Au, with a diameter of 75 nanometers and a distance of 110 nanometers between adjacent fingers, is intended to form a tetrameric coupling structure in the subsequent collapse process.

[0016] The antibody corresponding to (1-3)-β-D-glucan mentioned in step 2 is the stock solution. It is bound to the metal nanofinger array structure by low-temperature immersion and needs to be incubated for more than 12 hours, and repeated more than twice.

[0017] After incubation as described in step 2, the surface is washed multiple times to remove excess unbound antibodies.

[0018] The detection chip described in step 3 involves implanting a coupling structure into a bio-carrier chip to form a detection pool.

[0019] The serum and tears mentioned in step 4 are injected into and fill the entire built-in coupling structure of the biocarrier plate to ensure measurement in a liquid environment, and then left to stand for 15 minutes.

[0020] When performing the Raman spectroscopy measurement as described in step 5, it is necessary to focus on the surface of the gold-coupled structure to ensure that the measurement is of the bimolecular Raman fingerprint characteristic peaks of (1-3)-β-D-glucan and its antibody that are captured on the surface.

[0021] The Raman detection described in step 5 uses a 785 laser with an integration time of 1 second.

[0022] The (1-3)-β-D-glucan in the serum and tears mentioned in step 5 was analyzed using its 1134 cm⁻¹. -1 The intensity of the characteristic peak and the antibody characteristic peak at 1065 cm⁻¹ -1 The intensity ratio is used as a standard for quantitative detection.

[0023] The beneficial effects of this invention are:

[0024] 1. The fabrication of the detection chip involves combining Au flexible nanofinger with antibody incubation technology and embedding it at the bottom of the biocarrier sheet to form a pool for liquid detection.

[0025] 2. Since serum and tears are used as test samples, no further processing is required, and the measurement is carried out in a liquid environment. This allows for rapid and accurate qualitative and quantitative detection of the complex fingerprint of (1-3)-β-D-glucan molecules in serum and tears. The detection time is 15 minutes, which meets the clinical requirements for the detection of invasive fungal diseases.

[0026] 3. The surface-enhanced Raman substrate of the Au nanofinger / antibody / Au nanofinger coupling structure described in this invention has a wide range of applications and can be used to detect other biomolecules. Attached Figure Description

[0027] Figure 1 This diagram illustrates a method for detecting (1-3)-β-D-glucan, a biomarker for invasive fungal infections, based on an Au flexible nanofinger array structure. First, Au flexible nanofingers are immersed in a (1-3)-β-D-glucan antibody solution. Then, through a collapse effect, a coupled structure of Au nanofinger / antibody / Au nanofinger is formed, with the (1-3)-β-D-glucan antibody serving as a spacer layer. Finally, a serum or tear sample solution containing (1-3)-β-D-glucan molecules is dripped into the coupled structure. The antibody specifically captures (1-3)-β-D-glucan and localizes it in the coupled electromagnetic field, achieving high sensitivity and selectivity in SERS measurement.

[0028] Figure 2Scanning electron microscope (SEM) images of tetramer and multimer coupled structures formed by an ordered array of Au flexible nanofingers prepared by nanoimprinting after modification with (1-3)-β-D-glucan antibody solution and drying.

[0029] Figure 3 In Figure a, (a) shows the Raman spectra of the (1-3)-β-D-glucan antibody and (1-3)-β-D-glucan after they were dropped onto a gold film. This allows us to identify the characteristic peaks of both and pinpoint their differences. Figure b shows the Raman spectra of the antibody (green line), pure antibody (blue line), and Au nanofinger (black line) in the Au nanofinger / antibody / Au nanofinger coupled structure. The results show that the Au nanofinger has no Raman signal, but after the formation of the Au nanofinger / antibody / Au nanofinger coupled structure, the measured Raman signal is consistent with that of the pure antibody, indicating that the antibody was successfully incubated onto the surface of the Au finger.

[0030] Figure 4 Image a shows the Raman spectra of a detection chip filled with an Au nanofinger / antibody / Au nanofinger coupling structure, obtained from a pure solution of (1-3)-β-D-glucan (green line), patient serum (red line, clinically proven to be from an invasive fungal infection), and normal healthy human serum (blue line). The results show that the signal of (1-3)-β-D-glucan can be successfully detected in serum; image b shows the concentration gradient of the pure (1-3)-β-D-glucan solution as a function of the intensity of the glucan characteristic peak (selected at 1134 cm⁻¹). -1 ) / Antibody characteristic peak (select 1065cm) -1 The Raman peak intensity ratio of the concentration ranged from 1 pg / mL to 100 pg / mL, and the concentrations showed a linear distribution.

[0031] Figure 5 Image a shows the Raman spectrum of a detection chip filled with an Au nanofinger / antibody / Au nanofinger coupling structure in a patient's tear sample (clinically proven to be an invasive fungal infection). The results indicate that a relatively clear Raman characteristic peak of (1-3)-β-D-glucan in tears can be obtained after a 15-minute settling time, which can be used for fingerprint identification. Image b shows the Raman signal mapping of (1-3)-β-D-glucan obtained by Raman surface scanning after 15 minutes of settling (selecting 1134 cm⁻¹). -1 (Graphing was performed), with a scanning range of 100 μm × 100 μm, scanning one point every 10 μm, and a scanning time of 0.5 seconds for each point. The results showed that (1-3)-β-D-glucan was detected in most areas after standing for 15 minutes. Detailed Implementation

[0032] Example 1

[0033] A method for detecting (1-3)-β-D-glucan molecules in patient serum based on surface-enhanced Raman scattering technology includes the following steps:

[0034] Step 1: On a silicon substrate, a flexible Au nanofinger ordered array structure is prepared using nanoimprinting, Au thin film deposition, and reactive ion beam etching. The finger pillars are made of UV imprinting adhesive, with a deposited Au layer on top. The height of a single nanofinger is 350 nm, including a 300 nm polymer pillar and a 50 nm Au layer on top, with a diameter of 75 nm and a distance of 110 nm between adjacent fingers.

[0035] Step 2: Remove the (1-3)-β-D-glucan aptamer from the -20℃ freezer, thaw it at room temperature, and then heat it in a water bath at 37℃ for 30 min.

[0036] Step 3: The ordered array structure of flexible Au nanofinger obtained in Step 1 was immersed in the antibody solution corresponding to (1-3)-β-D-glucan obtained in Step 2 and incubated at 4 °C for 12 h to allow the antibody molecules in the solution to adsorb and connect with the Au surface. After the incubation time, it was removed and dried. During the drying process, the micro-surface tension generated by solvent evaporation caused the Au nanofingers to collapse and stick together, forming a tetrameric Au nanofinger / antibody / Au nanofinger coupling structure based on antibody molecules as gaps. See details. Figure 2 As shown; antibody molecules are distributed on the surface of the Au layer, eventually forming a molecular capture system. The above incubation process is repeated twice.

[0037] Step 4: Aptamer and (1-3)-β-D-glucan were dropped onto two 25nm thick gold films respectively, and Raman spectroscopy was performed to calibrate the characteristic peaks. Specifically, as follows... Figure 3 As shown, the peak position of (1-3)-β-D-glucan is at 1134 cm⁻¹. -1 877cm -1 Place, such as Figure 3 (a) Red area.

[0038] Step 5: Raman spectroscopy was performed on the Au nanofinger / antibody / Au nanofinger coupled structure obtained in Step 3. The Raman spectrum was compared with that of a clean gold nanofinger array and the Raman spectrum of (1-3)-β-D-glucan aptamers dropped onto a gold film. The resulting Raman spectra are shown in the figure. Figure 3 As shown in (b), the (1-3)-β-D-glucan aptamer has been uniformly incubated onto the gold nanofinger array.

[0039] Step 6: In the Au nanofinger array structure chip obtained in Step 3, inject a pure (1-3)-β-D-glucan solution with a concentration of 1-100 pg / mL. Raman detection is performed in a liquid environment using a water microscope lens. First, it is confirmed that the Raman characteristic signal of (1-3)-β-D-glucan can be detected after 15 minutes. Then, a 1134 cm⁻¹ dextran solution is used. -1 The intensity of the characteristic peak and the antibody characteristic peak at 1065 cm⁻¹ -1 The ratio of intensity is used to establish a curve for quantitative measurement; see details below. Figure 4 As shown in (b).

[0040] Step 7: Patient serum and healthy human serum, which had undergone centrifugation and lysis treatment, were injected into the Au nanofinger array structure chip obtained in Step 3. After 15 minutes, Raman detection was performed in a liquid environment using a water microscope. The excitation wavelength was 785 nm, and the excitation time was 1 second. By comparing the Raman fingerprint characteristic peaks with those of (1-3)-β-D-glucan and its antibody, it was found that the Raman fingerprint characteristic peaks of (1-3)-β-D-glucan could be captured and obtained from complex serum, but no Raman peaks were found for other protein molecules. This confirms that the specific detection of (1-3)-β-D-glucan can be achieved. See details below. Figure 4 As shown in (a).

[0041] The detection advantages of this embodiment are:

[0042] I. The gap in the coupling structure is determined by the sub-nanometer size of (1-3)-β-D-glucan.

[0043] Second, it can quickly replicate samples over a large area.

[0044] Third, it has extremely high repeatability and stability.

[0045] IV. Utilizing the binding of antibodies and antigens to achieve specific detection of biomolecules.

[0046] Fifth, it shortened the detection time of (1-3)-β-D-glucan in serum.

[0047] Example 2

[0048] A method for detecting (1-3)-β-D-glucan molecules in patient tears based on surface-enhanced Raman scattering technology includes the following steps:

[0049] Step 1: Patient tears that had undergone centrifugation and lysis were injected into a gold nanofinger array chip. After 15 minutes, Raman surface distribution scanning was performed in the liquid environment using a water microscope lens. The excitation wavelength was 785 nm, the scanning range was 100 μm × 100 μm, and a point was scanned every 10 μm, with a scanning time of 0.5 seconds per point. The Raman signal mapping of (1-3)-β-D-glucan obtained by Raman surface scanning (selected at 1134 cm⁻¹) is shown. -1 (Drawing), such as Figure 5 As shown in -b. The Raman spectra of representative red points extracted from the 0-minute and 15-minute surface scans were compared, as shown... Figure 5 As shown in -a, the results indicate that by comparing the Raman fingerprint characteristic peaks of (1-3)-β-D-glucan and its antibody, it was found that the Raman fingerprint characteristic peaks of (1-3)-β-D-glucan could be captured and obtained from complex tears, but there were no Raman peaks of other protein molecules, thus confirming that the specific detection of (1-3)-β-D-glucan can be achieved.

[0050] The detection advantages of this embodiment are:

[0051] I. The gap in the coupling structure is determined by the sub-nanometer size of (1-3)-β-D-glucan.

[0052] Second, it can quickly replicate samples over a large area.

[0053] Third, it has extremely high repeatability and stability.

[0054] IV. Utilizing the binding of antibodies and antigens to achieve specific detection of biomolecules.

[0055] Fifth, it shortened the detection time of (1-3)-β-D-glucan in tears.

Claims

1. A method for detecting (1-3)-β-D-glucan molecules based on surface-enhanced Raman scattering (SERS), characterized in that... Includes the following steps: Step 1: On a silicon substrate, a flexible Au nanofinger ordered array structure is prepared by nanoimprinting, Au thin film deposition and reactive ion beam etching, wherein the finger pillars are made of UV imprinting adhesive and Au layer is deposited on top; Step 2: The ordered array structure of flexible Au nanofinger obtained in Step 1 was immersed in the antibody solution corresponding to (1-3)-β-D-glucan and incubated in a refrigerator at 4 ℃ for 12 h, so that the antibody molecules in the solution were adsorbed and connected to the Au surface. After the incubation time was over, it was taken out and dried. During the drying process, the micro-surface tension generated by solvent evaporation caused the Au nanofingers to collapse and stick together, forming a tetrameric Au nanofinger / antibody / Au nanofinger coupling structure based on antibody molecules as gaps. The antibody molecules are distributed on the surface of the Au layer, and finally a molecular capture system is formed. The above incubation process was repeated twice. Step 3: Implant the tetrameric Au nanofinger / antibody / Au nanofinger coupling structure obtained in Step 2 into the biocarrier chip to form a detection chip; Step 4: Fill the tetrameric Au nanofinger / antibody / Au nanofinger coupled structure detection chip obtained in Step 3 with filtered and lysed complex serum and tears containing (1-3)-β-D-glucan, and let it stand for 15 minutes or more. Step 5: Raman measurement is performed on the detection chip filled with liquid serum or tears from Step 4. The Raman spectrometer is focused onto the chip surface in the liquid, the excitation wavelength is 785 nm, and the time is 1 second. A bimolecular Raman spectrum containing only (1-3)-β-D-glucan and its corresponding antibody is obtained. Specific identification is performed through the fingerprint characteristic peaks of both, and the results are obtained through the 1134 cm⁻¹ peak. -1 and 1065cm -1 The Raman peak intensity ratio is used as the standard for quantitative detection.

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